Survival of Lactobacillus casei strain Shirota in the intestines of healthy Chinese adults

Lactobacillus casei strain Shirota (LcS) is a widely used probiotic strain with health benefits. In this study, the survival of LcS in the intestines of healthy Chinese adults was assessed and the effects of LcS on stool consistency, stool SCFAs and intestinal microbiota evaluated. Subjects consumed 100 mL per day of a probiotic beverage containing 1.0 × 10⁸ CFU/mL of LcS for 14 days. LcS were enumerated using a culture method and the colony identity confirmed by ELISA. Fourteen days after ingestion, the amount of LcS recovered from fecal samples was between 6.86 ± 0.80 and 7.17 ± 0.57 Log₁₀ CFU/g of feces (mean ± SD). The intestinal microbiotas were analyzed by denaturing gradient gel electrophoresis. Principal component analysis showed that consuming LcS significantly changed fecal microbiota profiles. According to redundancy analysis, the amounts of 25 bacterial strains were significantly correlated with LcS intake (P < 0.05), 11 of them positively and fourteen negatively. Concentrations of acetic acid and propionic acid in feces were significantly lower during the ingestion period than during the baseline period (P < 0.05). These results confirm that LcS can survive passage through the gastrointestinal tract of Chinese people; however, they were found to have little ability to persist once their consumption had ceased. Furthermore, consumption of probiotic beverages containing LcS can modulate the composition of the intestinal microbiota on a long‐term basis, resulting in decreased concentrations of SCFAs in the gut.     

In vitro and in vivo inhibition of Helicobacter pylori by Lactobacillus casei strain Shirota

We studied the potential inhibitory effect of Lactobacillus casei strain Shirota (from the fermented milk product Yakult [Yakult Ltd., Tokyo, Japan]) on Helicobacter pylori by using (i) in vitro inhibition assays with H. pylori SS1 (Sydney strain 1) and nine H. pylori clinical isolates and (ii) the in vivo H. pylori SS1 mouse model of infection over a period of 9 months. In vitro activity against H. pylori SS1 and all of the clinical isolates was observed in the presence of viable L. casei strain Shirota cells but not in the cell-free culture supernatant, although there was profound inhibition of urease activity. In vivo experiments were performed by oral administration of L. casei strain Shirota in the water supply over a period of 9 months to 6-week-old C57BL/6 mice previously infected with H. pylori SS1 (study group; n = 25). Appropriate control groups of H. pylori-infected but untreated animals (n = 25) and uninfected animals given L. casei strain Shirota (n = 25) also were included in the study. H. pylori colonization and development of gastritis were assessed at 1, 2, 3, 6, and 9 months postinfection. A significant reduction in the levels of H. pylori colonization was observed in the antrum and body mucosa in vivo in the lactobacillus-treated study group, as assessed by viable cultures, compared to the levels in the H. pylori-infected control group. This reduction was accompanied by a significant decline in the associated chronic and active gastric mucosal inflammation observed at each time point throughout the observation period. A trend toward a decrease in the anti-H. pylori immunoglobulin G response was measured in the serum of the animals treated with lactobacillus, although this decrease was not significant.     

In Vitro and In Vivo Inhibition of Helicobacter pylori by Lactobacillus casei Strain Shirota

We studied the potential inhibitory effect of Lactobacillus casei strain Shirota (from the fermented milk product Yakult [Yakult Ltd., Tokyo, Japan]) on Helicobacter pylori by using (i) in vitro inhibition assays with H. pylori SS1 (Sydney strain 1) and nine H. pylori clinical isolates and (ii) the in vivo H. pylori SS1 mouse model of infection over a period of 9 months. In vitro activity against H. pylori SS1 and all of the clinical isolates was observed in the presence of viable L. casei strain Shirota cells but not in the cell-free culture supernatant, although there was profound inhibition of urease activity. In vivo experiments were performed by oral administration of L. casei strain Shirota in the water supply over a period of 9 months to 6-week-old C57BL/6 mice previously infected with H. pylori SS1 (study group; n = 25). Appropriate control groups of H. pylori-infected but untreated animals (n = 25) and uninfected animals given L. casei strain Shirota (n = 25) also were included in the study. H. pylori colonization and development of gastritis were assessed at 1, 2, 3, 6, and 9 months postinfection. A significant reduction in the levels of H. pylori colonization was observed in the antrum and body mucosa in vivo in the lactobacillus-treated study group, as assessed by viable cultures, compared to the levels in the H. pylori-infected control group. This reduction was accompanied by a significant decline in the associated chronic and active gastric mucosal inflammation observed at each time point throughout the observation period. A trend toward a decrease in the anti-H. pylori immunoglobulin G response was measured in the serum of the animals treated with lactobacillus, although this decrease was not significant.     

Effect of Different Antibiotics and Non-Steroidal Anti-Inflammatory Drugs on the Growth of Lactobacillus casei Shirota

The purpose of this study was to investigate whether some non-steroidal anti-inflammatory drugs (NSAIDs) could cause inhibition of the growth of Lactobacillus casei Shirota (LcS) or whether this microorganism is able to use some of them as the sole carbon source, considering that the simultaneous consumption of NSAIDs and a dairy drink fermented with LcS could help to prevent the appearance or improve the healing of gastric ulcers. Acetylsalicylic acid (ASA), sodium acetylsalicylate (SAS), acetaminophen, sodium naproxen, and sodium ibuprofen were added as the sole carbon source to a basal medium and tested for biodegradation by LcS. The same NSAIDs were added in different concentrations to disks and plated on MRS Agar to test the possible inhibitory effect of these compounds on LcS. Also, the resistance of LcS to 12 different antibiotics was studied on MRS agar. None of the NSAIDs tested could be used by LcS as the sole carbon source at the assayed concentrations. In the case of the disk diffusion method, sodium naproxen showed inhibition zones for the 500-μg disks and sodium ibuprofen was inhibitory for the 250- and 500-μg disks. However, when the macrobroth dilution method was used, the growth of LcS was inhibited by ASA, SAS, acetaminophen, and sodium ibuprofen. This strain showed resistance to the antibiotics sulfamethoxazole with trimethoprim, pefloxacin, and gentamicin. This is the first study on the effect of NSAIDs on probiotic bacteria. The results of the biodegradation test indicate that the simultaneous consumption of NSAIDs and a dairy beverage with LcS is not likely to change the bioavailability of the drugs.     

Beneficial effect of oral administration of Lactobacillus casei strain Shirota on insulin resistance in diet-induced obesity mice

Aims: This study aimed at determining whether oral administration of a probiotic strain, Lactobacillus casei strain Shirota (LcS), can improve insulin resistance, which is the underlying cause of obesity‐associated metabolic abnormalities, in diet‐induced obesity (DIO) mice. Methods and Results: DIO mice were fed a high‐fat diet without or with 0·05% LcS for 4 weeks and then subjected to an insulin tolerance test (ITT) or oral glucose tolerance test (OGTT). Oral administration of LcS not only accelerated the reduction in plasma glucose levels during the ITT, but also reduced the elevation of plasma glucose levels during the OGTT. In addition, plasma levels of lipopolysaccharide‐binding protein (LBP), which is a marker of endotoxaemia, were augmented in the murine models of obese DIO, ob/ob, db/db and KK‐A^y and compared to those of lean mice. LcS treatment suppressed the elevation of plasma LBP levels in DIO mice, but did not affect intra‐abdominal fat weight. Conclusions: LcS improves insulin resistance and glucose intolerance in DIO mice. The reduction in endotoxaemia, but not intra‐abdominal fat, may contribute to the beneficial effects of LcS. Significance and Impact of the Study: This study suggests that LcS has the potential to prevent obesity‐associated metabolic abnormalities by improving insulin resistance.     

Release studies of Lactobacillus casei strain Shirota from chitosan-coated alginate-starch microcapsules in ex vivo porcine gastrointestinal contents

AIMS: To investigate the release characteristics of Lactobacillus casei strain Shirota (LCS) from chitosan-coated alginate-starch (CCAS) capsule in different regions of ex vivo porcine gastrointestinal (GI) contents. METHODS AND RESULTS: A 0.1 g of CCAS encapsulated bacteria (containing c. 10(8) CFU of LCS) were incubated in different sections of ex vivo porcine GI contents (10 ml) anaerobically at 37 degrees C. Samples were taken from different GI contents at different time intervals (up to 24 h) and estimated for the release of LCS from capsules. There was almost a complete release (1.1 x 10(8) CFU) of LCS from capsules within 8 h of incubation in ileal contents, while it took nearly 12 h to release the completely encapsulated bacteria in colon content under similar conditions. There was only a partial release of encapsulated LCS, incubated in duodenal and jejunal contents, while there was no significant (P > 0.05) release of encapsulated bacteria in gastric contents even after 24 h of incubation. CONCLUSIONS: The capsules were able to release viable probiotic cells completely in ex vivo porcine ileal and colon contents. SIGNIFICANCE AND IMPACT OF THE STUDY: CCAS capsule can be an effective way of protecting probiotic bacteria from adverse gastric conditions and delivering viable bacteria to the host intestinal systems.     

Effects of Lactobacillus casei Shirota, Bifidobacterium breve, and oligofructose-enriched inulin on colonic nitrogen-protein metabolism in healthy humans

Pre- and/or probiotics can cause changes in the ecological balance of intestinal microbiota and hence influence microbial metabolic activities. In the present study, the influence of oligofructose-enriched inulin (OF-IN), Lactobacillus casei Shirota, and Bifidobacterium breve Yakult on the colonic fate of NH3 and p-cresol was investigated. A randomized, placebo-controlled, crossover study was performed in 20 healthy volunteers to evaluate the influence of short- and long-term administration of OF-IN, L. casei Shirota, B. breve Yakult, and the synbiotic L. casei Shirota + OF-IN. The lactose[15N,15N]ureide biomarker was used to study the colonic fate of NH3. Urine and fecal samples were analyzed for 15N content by combustion-isotope ratio mass spectrometery and for p-cresol content by gas chromatography-mass spectrometry. RT-PCR was applied to determine the levels of total bifidobacteria. Both short- and long-term administration of OF-IN resulted in significantly decreased urinary p-cresol and 15N content. The reduction of urinary 15N excretion after short-term OF-IN intake was accompanied by a significant increase in the 15N content of the fecal bacterial fraction. However, this effect was not observed after long-term OF-IN intake. In addition, RT-PCR results indicated a significant increase in total fecal bifidobacteria after long-term OF-IN intake. Long-term L. casei Shirota and B. breve Yakult intake showed a tendency to decrease urinary 15N excretion, whereas a significant decrease was noted in p-cresol excretion. In conclusion, dietary addition of OF-IN, L. casei Shirota, and B. breve Yakult results in a favorable effect on colonic NH3 and p-cresol metabolism, which, in the case of OF-IN, was accompanied by an increase in total fecal bifidobacteria.     

A kinetic study of wild-type and mutant dihydrofolate reductases from Lactobacillus casei

A kinetic scheme is presented for Lactobacillus casei dihydrofolate reductase that predicts steady-state kinetic parameters. This scheme was derived from measuring association and dissociation rate constants and pre-steady-state transients by using stopped-flow fluorescence and absorbance spectroscopy. Two major features of this kinetic scheme are the following: (i) product dissociation is the rate-limiting step for steady-state turnover at low pH and follows a specific, preferred pathway in which tetrahydrofolate (H4F) dissociation occurs after NADPH replaces NADP+ in the ternary complex; (ii) the rate constant for hydride transfer from NADPH to dihydrofolate (H2F) is rapid (khyd = 430 s-1), favorable (Keq = 290), and pH dependent (pKa = 6.0), reflecting ionization of a single group. Not only is this scheme identical in form with the Escherichia coli kinetic scheme [Fierke et al. (1987) Biochemistry 26, 4085] but moreover none of the rate constants vary by more than 40-fold despite there being less than 30% amino acid homology between the two enzymes. This similarity is consistent with their overall structural congruence. The role of Trp-21 of L. casei dihydrofolate reductase in binding and catalysis was probed by amino acid substitution. Trp-21, a strictly conserved residue near both the folate and coenzyme binding sites, was replaced by leucine. Two major effects of this substitution are on (i) the rate constant for hydride transfer which decreases 100-fold, becoming the rate-limiting step in steady-state turnover, and (ii) the affinities for NADPH and NADP+ which decrease by approximately 3.5 and approximately 0.5 kcal mol-1, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Insertion of bacteriophage phiFSW into the chromosome of Lactobacillus casei strain Shirota (S-1): characterization of the attachment sites and the integrase gene

The integrase gene (int) on the genome of φFSW, which is a temperate bacteriophage of Lactobacillus casei strain Shirota (formerly denoted as S-1), and the four attachment sites on the genomes of the phage and its host were characterized by sequencing. The φFSW integrase was found to belong to the integrase family of site-specific tyrosine recombinase. The attachment sites shared a 40bp common core within which an integrative site-specific recombination occurs. The common core was flanked on one side by an additional segment of high sequence similarity. An integration plasmid, consisting of int, the phage attachment site (attP), and a selectable marker, inserted stably into the bacterial attachment site (attB) within the common core, as did the complete prophage genome at a frequency of more than 10(3)/microg of plasmid DNA. This plasmid was used as a test system for a preliminary mutational analysis of int and attP. The attB common core was located within and near the end of an open reading frame that appears to encode a homolog to glucose 6-phosphate isomerase, an enzyme of the glycolytic pathway. It is unlikely that the prophage integration inactivates this protein, since a change of only the C-terminal amino acid is predicted because of the sequence similarity between attP and attB.     

Effects of a fermented milk drink containing Lactobacillus casei strain Shirota on the immune system in healthy human subjects

Nine healthy volunteers drank fermented milk containing 4 x 10(10) live cells of Lactobacillus casei strain Shirota daily for 3 weeks, and their NK activity and other immunological functions were measured. NK activity significantly increased (p<0.01) 3 weeks after the start of intake and remained elevated for the next 3 weeks. The effect was particularly prominent in low-NK-individuals.     

Effects of a Fermented Milk Drink Containing Lactobacillus casei Strain Shirota on the Immune System in Healthy Human Subjects

Nine healthy volunteers drank fermented milk containing 4×10¹⁰ live cells of Lactobacillus casei strain Shirota daily for 3 weeks, and their NK activity and other immunological functions were measured. NK activity significantly increased (p<0.01) 3 weeks after the start of intake and remained elevated for the next 3 weeks. The effect was particularly prominent in low-NK-individuals.     

Citrate metabolism in Lactobacillus casei and Lactobacillus plantarum

Information on the factors influencing citrate metabolism in lactobacilli is limited and could be useful in understanding the growth of lactobacilli in ripening cheese. Citrate was not used as an energy source by either Lactobacillus casei ATCC 393 or Lact. plantarum 1919 and did not affect the growth rate when co-metabolized with glucose or galactose. In growing cells, metabolism of citrate was minimal at pH 6 but significant at pH 4·5 and was greater in cells co-metabolizing galactose than in those co-metabolizing glucose or lactose. In non-growing cells, optimum utilization of citrate also occurred at pH 4·5 and was not increased substantially by the presence of fermentable sugars. In both growing and non-growing cells, acetate and acetoin were the major products of citrate metabolism; pyruvate was also produced by non-growing cells and was transformed to acetoin once the citrate was exhausted. Citrate was metabolized more rapidly than sugar by non-growing cells; the reverse was true of growing cells. Citrate metabolism by Lact. plantarum 1919 and Lact. casei ATCC 393 increased six- and 22-fold, respectively, when the cells were pre-grown on galactose plus citrate than when pre-grown on galactose only. This was probably due to induction of citrate lyase by growth on citrate plus sugar. These results imply that lactobacilli, if present in large enough numbers, can metabolize citrate in ripening cheese in the absence of an energy source.     

Regulation of lactose-phosphoenolpyruvate-dependent phosphotransferase system and beta-D-phosphogalactoside galactohydrolase activities in Lactobacillus casei.

The lactose-phosphoenolpyruvate-dependent phosphotransferase system (lac-PTS) and beta-D-phosphogalactoside galactohydrolase (P-beta-gal) mediate the metabolism of lactose by Lactobacillus casei. Starved cells of L. casei contained a high intracellular concentration of phosphoenolpyruvate, and this endogenous energy reserve facilitated characterization of phosphotransferase system activities in physiologically intact cells. Data obtained from transport studies with whole cells and from in vitro phosphotransferase system assays with permeabilized cells revealed that the lac-PTS had a high affinity for beta-galactosides (e.g., lactose, lactulose, lactobionic acid, and arabinosyl-beta-D-galactoside). lac-PTS and P-beta-gal activities were determined in wild-type strains and strains defective in the glucose-phosphoenolpyruvate-dependent phosphotransferase system after growth on various sugars and in the presence of potential inducers. We found that (i) the lac genes (i.e., the genes coding for the lac-PTS proteins and P-beta-gal) were induced by metabolizable and non-metabolizable beta-galactosides (presumably acting as their phosphorylated derivatives), (ii) galactose 6-phosphate was not an inducer in most strains, (iii) the ratio of lac-PTS activity to P-beta-gal activity in a given strain was not constant, and (iv) inhibition of lac gene expression during growth on glucose was a consequence of glucose-phosphoenolpyruvate-dependent phosphotransferase system-mediated inducer exclusion, repressive effects of a functional glucose-phosphoenolpyruvate-dependent phosphotransferase system and glucose-derived metabolites. The expression of the lac-PTS structural genes and the expression of the P-beta-gal gene are independently regulated and may be subject to both positive control and negative control.     

Determination of the kinetic constants of fructose transport and phosphorylation in the perfused rat liver

The kinetics of fructose uptake was determined in perfused rat liver during steady-state fructose elimination. On the basis of the corresponding values of fructose concentration in the affluent and in the effluent medium, and the fructose and ATP concentration in biopsies, the kinetics of membrane transport and intracellular phosphorylation in the intact organ was calculated according to a model system. Carrier-mediated fructose transport has a high $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ (67 mM) and $\text{V}$ (30 μmoles · min^?1 ·g^?1). The calculated kinetic constants of the intracellular phosphorylation were compared with values obtained with an acid-treated rat liver high speed supernatant (values given in parentheses). $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ with fructose 1.0 mM (0.7 mM), $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ with ATP 0.54 mM (0.37 mM), $\text{V}$ 10.3 μmoles · min^?1 · g^?1 (10.1 μmoles · min^?1 · g^?1, calculated on the basis of the highest measured rate of fructose uptake correcting the ATP concentration to saturating values). The kinetics of fructose uptake reveals that at Physiological fructose concentrations the membrane transport limits the rate of fructose uptake, thus protecting the liver from severe depletion of adenine nucleotides.     

Protective effect of Lactobacillus casei strain Shirota against lethal infection with multi-drug resistant Salmonella enterica serovar Typhimurium DT104 in mice

Aims: The anti‐infectious activity of lactobacilli against multi‐drug resistant Salmonella enterica serovar Typhimurium DT104 (DT104) was examined in a murine model of an opportunistic antibiotic‐induced infection. Methods and Results: Explosive intestinal growth and subsequent lethal extra‐intestinal translocation after oral infection with DT104 during fosfomycin (FOM) administration was significantly inhibited by continuous oral administration of Lactobacillus casei strain Shirota (LcS), which is naturally resistant to FOM, at a dose of 10⁸ colony‐forming units per mouse daily to mice. Comparison of the anti‐Salmonella activity of several Lactobacillus type strains with natural resistance to FOM revealed that Lactobacillus brevis ATCC 14869^T, Lactobacillus plantarum ATCC 14917^T, Lactobacillus reuteri JCM 1112^T, Lactobacillus rhamnosus ATCC 7469^T and Lactobacillus salivarius ATCC 11741^T conferred no activity even when they obtained the high population levels almost similar to those of the effective strains such as LcS, Lact. casei ATCC 334^T and Lactobacillus zeae ATCC 15820^T. The increase in concentration of organic acids and maintenance of the lower pH in the intestine because of Lactobacillus colonization were correlated with the anti‐infectious activity. Moreover, heat‐killed LcS was not protective against the infection, suggesting that the metabolic activity of lactobacilli is important for the anti‐infectious activity. Conclusion: These results suggest that certain lactobacilli in combination with antibiotics may be useful for prophylaxis against opportunistic intestinal infections by multi‐drug resistant pathogens, such as DT104. Significance and Impact of the Study: Antibiotics such as FOM disrupt the metabolic activity of the intestinal microbiota that produce organic acids, and that only probiotic strains that are metabolically active in vivo should be selected to prevent intestinal infection when used clinically in combination with certain antibiotics.     

Effect of an oral administration of Lactobacillus casei strain Shirota on the natural killer activity of blood mononuclear cells in aged mice

The natural killer (NK) activity of blood mononuclear cells and splenocytes in aged mice fed on a diet containing Lactobacillus casei strain Shirota (LcS group) was significantly (P < 0.01) higher than that in mice fed on a diet without LcS. In the LcS group, there was a significant positive correlation (r = 0.63, P < 0.01) between the NK activity of blood mononuclear cells and the NK activity of splenocytes.